Oxygen switches: Refueling for cancer radiotherapy

Radiotherapy remains the major therapeutic intervention for tumor patients. However, the hypoxic tumor microenvironment leads to treatment resistance. Recently, a burgeoning number of nano-radiosensitizers designed to increase the oxygen concentration in tumors were reported. These nano radiosensitizers served as oxygen carriers, oxygen generators, and even sustained oxygen pumps, attracting increased research interest. In this review, we focus on the novel oxygen-enrich nano radiosensitizers, which we call oxygen switches, and highlight their influence in radiotherapy through different strategies. Physical strategies-based oxygen switches carried O2 into the tumor via their high oxygen capacity. The chemical reactions to generate O2 in situ were triggered by chemical strategies-based oxygen switches. Biological strategies-based oxygen switches regulated tumor metabolism, remodeled tumor vasculature, and even introduced microorganisms-mediated photosynthesis for long-lasting hypoxia alleviating. Moreover, the challenges and perspectives of oxygen switches-mediated oxygen-enrich radiotherapy were discussed.

distribution, reprogrammed energy metabolism, etc. Oxygenenriched strategies are necessary for refueling cancer radiotherapy.
To relieve hypoxia in the tumor microenvironment, various oxygen delivery strategies have been tested nowadays. Hyperbaric oxygen (HBO) inhalation cannot meet the need for radiotherapy, because the oxygen level only remained elevated for a short time (5). What is worse, combining erythropoietin treatment and radiotherapy for head and neck tumors resulted in significantly worse prognosis in patients (6). This phenomenon presumably ascribes to that squamous cell carcinoma tumor cells also express the erythropoietin receptor (7). Artificial blood including perfluorocarbon and hemoglobin-based oxygen-carrying solutions have been explored to increase the effectiveness of radiotherapy in rodent tumors (8). But contentious results for different doses and different schedules retarded clinical translation. A more precise and intelligent oxygen delivery strategy is needed.
To further improve the efficiency of radiotherapy, nano oxygen modulators have raised global attention. As oxygen serves as the fuel to fix radiation-induced DNA damage, radiotherapy could be boosted by evaluating oxygen concentration. In this review, a variety of agents introduced for oxygen modulation show magnifying effects for radiotherapy. Thus, we collectively refer to these nano agents as "Oxygen Switch", which allows precise and high performance in reshaping the hypoxia microenvironment ( Figure 1). The physical strategies including hemoglobin-based and perfluorocarbon-based oxygen carriers will be first introduced in this review, followed by a detailed discussion of chemical strategies including in situ H 2 O 2 catalytic decomposition and metallic oxide decomposition. Next, we highlighted novel biological strategies such as in-situ photosynthesis and tumor vasculature remodeling. The mechanism and applications of these "oxygen switches" for radiotherapy enhancement are also covered. Finally, the challenges and perspectives of oxygen switches utilized in radiotherapy are presented.

Physical strategies
To relieve the hypoxia in the tumor microenvironment and improve the efficacy of radiotherapy, delivering exogenous oxygen into the tumor site as a radiosensitizer was first explored in the 1930s (9). Physical strategies refer to directly delivering exogenous oxygen via oxygen carriers without chemical or biological reactions. Though physical only relieves hypoxia temporally, it is enough for the radiation process via precise tumor targeting. To date, A B FIGURE 1 The wrestling between tumor hypoxia and related alleviating strategies. (A) Tumor hypoxia remains established for multiple reasons, such as tumor cell proliferation, abnormal vascular distribution, reprogrammed energy metabolism, and so on. Thus, physical strategies, chemical strategies, and biological strategies are designed to relieve tumor hypoxia. (B) Oxygen switches increase O 2 supply or decrease O 2 consumption to combat treatment resistance to radiotherapy. perfluorocarbons (PFCs) and hemoglobin (Hb) or their derivatives are regarded as excellent oxygen carriers for their high oxygen capacity and favorable biocompatibility, and emerging physical strategies-based oxygen switches are established (Table 1).

PFC-based oxygen carriers
PFCs demonstrated an outstanding oxygen affinity due to their fluorine atoms in the carbon skeleton (21). Of the high biocompatibility and chemical stability, PFCs have been widely used in the clinic, such as in organ transplantation and ultrasound imaging. Once the high oxygen solubility of PFCs was found to enable tumor hypoxia alleviation, PFC-based oxygen carriers were developed to enhance radiotherapy (22). Wang et al. developed a tumor-targeted 1H, 1H-perfluorooctylamine-modified hyaluronic acid-coated perfluorocarbon oxygen carrier, O 2 @PFC@FHA (perfluorooctylamine-modified hyaluronic acid) (10). For the interaction between HA and CD44 and a large amount of oxygen dissolved in the PFC core, O 2 @PFC@FHA NPs not only improved the tumor targeting but also enabled more oxygen to reach the hypoxic area of the tumor. Moreover, the encapsulation of FHA reduced the leakage of oxygen in circulation and thereby alleviating tumor hypoxia and strengthening radiotherapy.
The platelet inhibition of PFC was neglected and might contribute to increasing red blood cell infiltration into tumors and improving oxygen supply. Zhou et al. screened all the perfluorocarbon compounds and found that perfluorotributylamine (PFTBA) processed the strongest platelet inhibition effect (17). Thus, the two-way O 2 delivery system PFTBA@HSA was established, which took advantage of the platelet inhibition effect of PFTBA ( Figure 2A). After the release of physical bound O 2 (first step), PFTBA inhibited platelet activation and led to an increase in red blood cell (RBC) infiltration, which delivered oxygen to the tumor as the second step. This work presented a simple but effective method to reverse the resistance of tumor hypoxia to radiotherapy.
For the prolonged blood circulation time, endogenous biomimetic methods were established (24). Gao et al. developed PFC@PLGA-RBC membrane (RBCM) NPs, in which the PFC core showed efficient loading of oxygen, as well as greatly prolonged blood circulation time because of the coating of RBCM (18). The treatment efficacy during radiotherapy was remarkably enhanced for the greatly relieved tumor hypoxia. Furthermore, Yu et al. reported a nano RBC is fabricated that replaces heme with perfluorodecalin (FDC) and coated with RBCM (20). This method enabled the delivery of FDC because it cannot be emulsified by any FDA-approved emulsifiers.

Hemoglobin-based oxygen carriers
For the reversible and inherent oxygen-carrying capability of hemoglobin (Hb), a higher Hb level helped improved the response rate of radiotherapy (25). However, free Hb could not be directly administrated for its poor stability, which breaks the redox homeostasis and causes a severe systemic reaction. Thus, physical encapsulation or chemical conjugation is conducted to overcome these shortcomings. Gao et al. reported that Hb and curcumin formed self-assembled nanoparticles (12). The self-assembly process was driven by hydrophobic forces and contributed to a higher cell absorption rate and lower cytotoxicity than free curcumin. Combined with the radiosensitivity of curcumin and the oxygen delivery of Hb, these nanoparticles effectively enhanced the radiotherapy for hepatocellular in vivo.
Hafnium (Hf), a high-Z radiosensitizer, coordinated with chlorin e6 (Ce6), and Hb was encapsulated to modulate the oxygen balance in the hypoxic TME by Wei et al. (14). The radioluminescence excited by Hf under X-ray irradiation was used to activate Ce6 for ROS generation by radiodynamic therapy (RDT). Such a multifunctional nanoplatform in the combination of oxygen supply and radiotherapy-RDT might provide a new therapeutic option for cancer eradication.

Chemical strategies
Oxygen can be generated in a large number of chemical reactions in nature. However, producing O 2 in vivo by decomposing oxygenated chemicals safely and steadily is not easy. Different kinds of oxygen switches were developed, including an oxygen catalyzer or being decomposed (Table 2).

Oxide-based oxygen generator
Early leakage of delivering exogenous O 2 into the tumor site remains a challenge for physical strategies. Thus, delivering the precursors of oxygen to the hypoxic area and generating O 2 in situ is attractive.
Chen et al. utilized CuO nanoparticles to generate O 2 under microwave irradiation in the tumor microenvironment (38). Decorated with MW sensitizer 1-butyl-3-methylimidazolium hexafluorophosphate (IL) and radiosensitizer of Quercetin (Qu), the mesoporous sandwich SiO 2 @ZrO 2 nanoparticles (SiO 2 @ZrO 2 NPs) persistently released oxygen under MW irradiation, which significantly increased the re-oxygenation ability of tumor cells. Due to the reshaping of the tumor microenvironment, a high inhibition rate of 98.62% was witnessed in the in vivo antitumor experiment.

Catalyzer-based oxygen generator
Due to the abnormal metabolism and redox homeostasis, a high level of H 2 O 2 is found in the tumor cells compared to normal cells. To generate O 2 via H 2 O 2 decomposition, catalase (CAT) and nanoenzyme-loaded oxygen switches were developed to enhance radiotherapy in situ.
CAT generates oxygen in situ but may be degraded in vivo due to the upregulated protease in the tumor. Zai et al. developed highly protease-resistant E. coli membrane vesicles (EMs) to contain CAT and thus relieve tumor hypoxia for a long time (3) ( Figure 2B). EMs demonstrated a higher CAT activity than free CAT even in the concentration of 100-fold protease. Combined with immune stimulation features, EMs maintained their hypoxia relief ability for a long time and enhanced radiotherapy. Song (48). CAT and H 2 O 2 were separately encapsulated within stealthy liposomes for a long-lasting effect in tumor reoxygenation enhancement. Furthermore, the relieved tumor hypoxia enhanced the therapeutic effects of radiotherapy and reversed the immunosuppressive tumor microenvironment. Combined with CTLA4 blockade, the radio-immunotherapy induced effective anti-tumor immune responses to destruct tumors.
Zhang et al. reported a nanoplatform based on poly(N-vinyl caprolactam) (PVCL) nanogels (NGs) co-loaded with gold (Au) and manganese dioxide (MnO 2 ) nanoparticles (NPs) for sensitized radiotherapy (28). MnO 2 displayed the CAT-mimic catalytic activity that decomposed H 2 O 2 to form O 2 and alleviate tumor hypoxia ( Figure 2C). Resulted Mn 2+ exerted a Fenton-like reaction to cause intracellular ROS and made the cells more susceptible to radiotherapy. Meanwhile, Au NPs and Mn (II) transformed from MnO 2 NPs guided the in vivo radiotherapy through dual mode CT/MR imaging.

Biological strategies
Though chemical strategies display a prolonged oxygen modulation capacity than physical strategies, the hypoxic environment reappears once the chemicals are exhausted. Since tumor hypoxia is the outcome of abnormal biological behavior of tumor cells, emerging biological strategies-based oxygen switches may provide exciting opportunities and should be highlighted (Table 3).

Oxygen switches to decrease oxygen consumption
To support accelerated cell proliferation, excessive oxygen consumed is one of the critical reasons for tumor hypoxia. Thus, metabolism regulation-based strategies that inhibit tumor aerobic respiration is promising.
As mitochondria refer to the energy house that consumes oxygen to generate energy for tumor growth, mitochondria-targeted interventions are believed to enhance radiotherapy. Gao et al. fabricated a mitochondria-targeted nano-platform via the integration of a selfassembled peptide and a positively charged cyclen (62). The positively charged cyclen anchored to the mitochondria and loaded lonidamine, which served as the energy stripper of cancer cells, inhibited energy metabolism and oxygen consumption. Combined with radiotherapy and endogenous apoptosis pathway, this mitochondria-targeted intervention led to tumor eradication in vivo.
Recently, therapeutic gases were attractive and found to exhibit regulation effects. Nitric oxide (NO), hydrogen sulfide (H 2 S), and carbon monoxide (CO) were utilized in tumor treatment for their therapeutic capacity. Duo et al. designed an irradiation-triggered NOrelease nano-prodrug to improve radiosensitization (62). Through the reaction of sodium nitroprusside and L-glutathione, high content of NO was released and thus inhibited cell respiration and oxygen consumption. Then O 2 accumulation improved the therapeutic outcomes under irradiation by generating more ROS in the tumor microenvironment. Besides, H 2 S was also employed as an oxygen switch to remodel oxygen metabolism by inhibiting cytochrome c oxidase activity in a high-Z metal ion-sensitized radiotherapy (54).

Oxygen switches to increase oxygen supply
One important reason for tumor hypoxia is the abnormal blood vessel. To obtain nutrients for growth and to metastasize, tumor blood vessels are leaky, tortuous, and saccular (63). Thus, Tumor vascular normalization is a promising method to increase blood perfusion and relieve tumor hypoxia. Wang et al. modified Au NPs with 8-hydroxyquinoline (HQ) to obtain AuHQ, which attenuated the expression of angiopoietin-2, vascular endothelial growth factor 2 (56). Moreover, AuHQ treatment increased pericyte coverage and modulated tumor leakage, which led to increased blood perfusion. Tumor vascular normalization not only alleviated tumor hypoxia but also contributed to an increased AuHQ accumulation. Ultimately, compared to Au NPs, the anti-tumor efficacy of radiotherapy was increased by 38% in the AuHQ group. Apart from chemical strategies, generating O 2 in situ could be achieved by biological strategies via a natural photosynthetic system (64, 65). Qiao et al. engineered RBCM to modify the algal surface and deliver this RBCM-Algae to the tumor to increase tumor oxygenation (58). With red light-induced photosynthesis, RBCM-Algae generated O 2 in situ and alleviated tumor hypoxia, and further led to reoxygenated radiotherapy. Cyanobacteria were also utilized for continuous photosynthetic oxygen evolution in a two-dimensional bismuthene platform with high-Z components, which demonstrated the photosynthetic hypoxia-alleviation capability and radiosensitization performance (59). These works exemplified the construction of microorganism-enabled oxygen switches for radiosensitizeraugmented radiotherapy.

Challenges and perspective
Given the central role of tumor hypoxia in the treatment resistance to radiotherapy, a various of oxygen switches were fabricated based on physical strategies, chemical strategies, and biological strategies. In this review, we summarized the oxygen switches designed for hypoxic-tumor radiotherapy. Physical oxygen switches served as a high-capacity carrier to deliver exogenous O 2 . Chemical oxygen switches triggered an in-situ reaction to generate O 2 in vivo. Biological oxygen switches reshaped the tumor microenvironment by regulating biological behavior or introducing microorganism-mediated photosynthesis. Over the past few years, prosperous designs gained much attention and gratifying results in pre-clinical experiments, but there remained several challenges to be addressed.
The first limitation refers to the efficacy of these oxygen switches. As known, the hypoxic tumor area is quite complex and exhibits steric heterogeneity. On the one hand, deepen understanding of the mechanism of tumor hypoxia and radiosensitization are necessary. On the other hand, oxygen switches have to improve the O 2 concentration continuously and accurately. Bio-mimic encapsulation was employed in oxygen switches to avoid early leakage and immune clearance (20). Stimulus-response (near infrared-triggered, irradiationtriggered, focused ultrasound-triggered, etc.) oxygen switch helped to achieve spatiotemporal specificity oxygen generation (62). As the penetration depth of NIR was limited, irradiation-triggered or focused ultrasound-triggered oxygen switch was worth further exploration (66). Moreover, novel in-situ microorganism-mediated photosynthesis could generate O 2 continuously. However, it is still a challenge to prolong the survival time of microorganisms.
Secondly, the biosafety and biocompatibility of these oxygen switches are of concern. Unexpected Hb exposure can cause severe side effects including blood clot formation, renal toxicity, and cardiovascular complications (67). The metal oxide may affect intracellular redox homeostasis and induce macromolecule dysfunction. Especially, CAT-mimic nano-enzyme enables activating the matrix metalloproteinases in the tumor tissue, which could lead to inflammation and even tumor metastasis (68). Introducing microorganism into a human may activate the unfavorable immune response and induces local microbiome disturbances (69). Finally, the coordination of oxygen switches and radiotherapy should be strengthened. The pharmacokinetics of oxygen switches should be tailored to the conduction of radiotherapy. Since radiotherapy is an oxygen-consumed intervention, the amount of oxygen generated from the oxygen switches has to be precisely measured to keep the balance of oxygen concentration and reach the best outcome. Furthermore, the combination of other therapeutic interventions such as photodynamic therapy and RDT may improve the utilization efficiency of oxygen.
Researchers paid much effort to the approach of oxygen-enriched radiotherapy. Nowadays, novel technologies are paving the path to more precise clinical medicine. Single-cell sequencing helped us understand how hypoxia is shaped and the evolutionary landscapes of tumor genomics (70). Nano-robots can follow a specified route and directionality under control, thus leading to a more precise oxygen delivery (71). Genome editing may improve the safety and oxygengenerating capability of microorganisms-mediated photosynthesis (72).
To be concluded, from the first attempt to combat hypoxia for radiotherapy to enhancement, multiple strategies have been developed to increase available oxygen. Though different strategies work on different principles, the efficacy of these oxygen switches has been widely recognized. However, there remain some obstacles before clinical translation. With a more in-depth understanding of tumor hypoxia, we should believe that better radiosensitized oxygen switches will emerge.

Author contributions
XL, HW, ZL, and FT wrote the manuscript. SL, JW, and WG revised the manuscript. All authors contributed to the article and approved the submitted version.